Nonaqueous capillary electrophoresis of imatinib mesylate and related substances

In the present study, nonaqueous capillary electrophoretic separation of imatinib mesylate (IM) and related substances, N-(5-amino-2-methylphenyl)-4-(3-pyridyl)-2-pyrimidinamine (PYA), N-(4-methyl-3-(4-(pyridin-3-yl)pyrimidin-2-ylamino)phenyl)-4-((piperazin-1-yl)methyl) benzamide (NDI) and 4-chloromethyl-N-(4-methyl-3-((4-(pyridin-3-yl) pyrimidin-2-yl) amino) phenyl) benzamide (CPB) was developed. The influential factors affecting separation, including type and concentration of the electrolyte, applied voltage, and buffer modifier were investigated. Baseline separation of the studied analytes was obtained using a buffer of 50 mM Tris and 50 mM methanesulfonic acid in methanol at a apparent pH (pH*) of 1.65. To enhance the sensitivity, large-volume sample stacking was employed for online concentration. The strongest analytical signal with a suitable separation was achieved when the injection time was 100 s. The linearity ranges of PYA and NDI were 0.100-2.50 μg mL(-1) , and that of CPB was 0.125-2.50 μg mL(-1) , with good coefficients (r(2) > 0.9948). The relative standard deviations of intra- and interday were satisfactory. Under the optimized conditions, seven batches of the synthesized samples were analyzed and CPB was detected in two batches. Owing to its simplicity, effectiveness, and low price, the developed method is promising for quality control of IM.     

Capillary electrophoresis for the determination of norfloxacin and tinidazole in pharmaceuticals with multi-response optimization

Capillary electrophoresis (CE) with UV photo-diode array detection technique was utilized to adopt new method for the analysis of norfloxacin and tinidazole in pharmaceuticals. Many CE aspects including separation, rapidity, sensitivity, ruggedness as well as the repeatability of qualitative and quantitative analyses were considered simultaneously for the purpose of optimization. Experimental design approach including factorial design and response surface methods were applied to optimize electrolyte concentration and the pH while injection time, voltage and column temperature were optimized using the univariate method. Successful results were obtained using 32.5 mmol l^−l phosphate electrolyte at pH 2.5, injection time 8.0 s, voltage 25 kV and column temperature 25 °C with detection at wavelength 301 nm. The analytical characteristics including recovery, intermediate precision, linear dynamic ranges, linearity and selectivity as well as limits of detection and quantification were demonstrated and the applicability to pharmaceuticals was studied. The newly provided method enjoys the advantages of CE over HPLC with respect to rapidity, ruggedness, simplicity in reagents and sample preparation as well as saving in reagents and samples.     

Capillary electrophoresis as a method for ozone determination in atmospheres

Capillary electrophoresis (CE) is used to quantify nitrate and nitrite extracted from nitrite-impregnated glass fiber filters (IGFF) that are used to monitor ozone in atmospheres. The amount of nitrate produced from conversion of nitrite in the filters is directly related to the amount of ozone passed over the filter. The limit of detection for ozone using the CE method is 1.17 ppml and the method is linear over two orders of magnitude. The effect of the excess nitrite in the IGFF on the limits of detection is discussed. Results from CE analyses of both active and passive filters are presented. The active filter results are compared to ion chromatographic analyses.     

Determination of sulfonate ester genotoxic impurities in imatinib mesylate by gas chromatography with mass spectrometry

Methyl methanesulfonate and ethyl methanesulfonate are potential genotoxic impurities in imatinib mesylate. In this work, a simple, sensitive, reliable, and fast gas chromatography with mass spectrometry method for the simultaneous determination of methyl methanesulfonate and ethyl methanesulfonate was developed and validated. Total analysis time was only 7 min. An n‐hexane/water solution was used to dissolve samples, and then extracted‐ion‐chromatogram mode was used to quantify methyl methanesulfonate and ethyl methanesulfonate. Calibration curves showed good linearity over the studied range for methyl methanesulfonate and ethyl methanesulfonate. The correlation coefficient of fit exceeded 0.999 for each impurity. The LOD and LOQ of methyl methanesulfonate and ethyl methanesulfonate were as low as 0.001 and 0.005 μg/mL, respectively, with RSDs of the peak area within 1.06–1.96%. Method accuracy was within 97.2–99.8% for methyl methanesulfonate and ethyl methanesulfonate. Therefore, this method can be used to quantify methyl methanesulfonate and ethyl methanesulfonate impurities at extremely low levels in imatinib mesylate.     

Solid-phase extraction and large-volume sample stacking with an electroosmotic flow pump in capillary electrophoresis for determination of methotrexate and its metabolites in human plasma

This paper describes approaches for large-volume sample stacking (LVSS) with an EOF pumpin CE for the determination of methotrexate (MTX) and its metabolites in human plasma. After pretreatment of plasma through a SPE cartridge, a large sample volume was loaded by hydrodynamic injection (3 psi, 70 s) into the capillary filled with phosphate buffer (70 mM, pH 6.0) containing 0.01% polyethylene oxide. Following removal of a large plug of sample matrix from the capillary using polarity switching (-25 kV), the separation of anionic analytes was subsequently performed without changing polarity again, achieving an improvement of sensitivity of around a 100-fold. The method was applied to therapeutic drug monitoring of MTX in one acute lymphoblastic leukemia patient. This study is one of very few applications showing the feasibility of LVSS in analysis of biological samples by CE.     

Decoration of silver nanoparticles on multi-walled carbon nanotubes: Investigation of its anti-acute leukemia property against acute myeloid leukemia and acute T cell leukemia

Recently, the development of carbon nanocomposites composed of carbon nanotubes and metal nanoparticles has attracted many interests because of their large potential for technological applications such as catalysts, sensors, biomedicine, and disinfection. In the present study, we described a simple chemistry method to synthesize multi-walled carbon nanotubes (MWCNTs) decorated with silver nanoparticles (Ag-NPs). Also, we investigated the antioxidant and anti-acute leukemia activities against acute myeloid leukemia and acute T cell leukemia cell lines. Ag NPs-MWCNTs were characterized and analyzed using common nanotechnology techniques including transmission electron microscopy (TEM), X-ray diffraction (XRD), energy dispersive X-ray spectroscopy (EDS), field emission-scanning electron microscopy (FE-SEM) and elemental mapping analysis. Also, 2,2-diphenyl-1-picrylhydrazyl (DPPH) test was performed to assess the antioxidant capacities of AgNO₃, MWCNTs, and Ag NPs-MWCNTs. It revealed similar antioxidant potentials for Ag NPs-MWCNTs and butylated hydroxytoluene. In MTT assay, Ag NPs-MWCNTs had very low cell viability (very high anti-acute leukemia properties) dose-dependently against 32D-FLT3-ITD (Acute myeloid leukemia cell line), Human HL-60/vcr (Acute myeloid leukemia cell line), Jurkat, Clone E6–1 (Acute T cell leukemia cell line), and J.RT3-T3.5 (Acute T cell leukemia cell line) without any cytotoxicity on human umbilical vein endothelial cell line (HUVEC; Normal cell line). In conclusion, the synthesized Ag NPs-MWCNTs revealed excellent antioxidant and cytotoxicity activities against acute myeloid leukemia and acute T cell leukemia cell lines in a dose depended manner. After confirming in the in vivo and clinical trials, these nanoparticles can be administrated in humans for the treatment of acute leukemia especially acute myeloid leukemia and acute T cell leukemia.     

Nonaqueous capillary electrophoresis method for the analysis of gleevec and its main metabolite in human urine

The viability of nonaqueous capillary electrophoresis (NACE) was investigated for determination of gleevec and its main metabolite in human urine using a fused-silica capillary. Baseline separation of the studied solutes was obtained using a nonaqueous solution composed of 12 mM ammonium acetate and 87.6 mM acetic acid in methanol-acetonitrile (ACN) (80:20, v:v) providing analysis time shorter than 3 min. Different aspects including stability of the solutions, linearity, accuracy and precision were studied in order to validate the method in the urine matrix. Detection limits of 24 microg L(-1) for gleevec and its metabolite were obtained. A robustness test of the method was carried out using the Plackett-Burman fractional factorial model with a matrix of 15 experiments. The developed method is simple, rapid and sensitive and has been used to determine gleveec and its metabolite at clinically relevant levels in human urine. Before NACE determination, a solid-phase extraction (SPE) procedure with a C18 cartridge was necessary. Real determination of these analytes in two patient urines were done.     

Capillary zone electrophoresis method for the simultaneous determination of cations in honey

A capillary electrophoresis system for the simultaneous determination of cations in honey samples has been developed. The complete separation and quantification of K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+, which represent more than 99% of the total content of cations in honey, can be achieved in 4 min with only a dilution and filtration of the honey sample. Electrolyte solution was composed by 10 mM imidazole as the carrier buffer and background absorbance provider and acetic acid as the complexing agent (pH 3.60). The running voltage was + 25 kV at 25 degree C. Indirect UV detection was achieved at 185 nm. Under the optimum conditions the detection limits ranged from 0.02 to 48.2 mg/kg and the quantification limits have ranged from 0.41 to 48.7 mg/kg. Precision data in honey samples analysed have shown repeatability and reproducibility RSD (%) lower than 2.84 and 6.62%, respectively. Recoveries of cations in honey samples analysed have ranged from 88.5 to 101.8%. These cations were identified by their relative migration times with regard to Ba2+ migration time used as reference standard and they were quantified by using an external standard calibration. Twenty-five honey samples were analysed to test the proposed method. Mean contents of 1.22 x 10(3), 93, 85, 54, 11, 1.9 and 2.3 mg/kg were found, respectively, for K+, Ca2+, Na+, Mg2+, Mn2+, Ni2+ and Li+ cations in analysed honeys. These results were similar than the obtained by other authors.     

Capillary electrophoresis method for determination of related substances in glutathione reduced drug substance

Summary A sensitive capillary electrophoretic method using low pH with direct UV detection has been developed to evaluate the impurity profile of reduced glutathione obtained from its production and purification. The method is characterized by high detection sensitivity and selectivity. Validation has been performed with model mixtures consisting of the main known related substances—oxidized glutathione, glutamylcystein, cysteinylglycine and cysteine. The results from this study show that with respect to quantification criteria the method is acceptable for routine control of reduced glutathione for pharmaceutical application.     

Capillary electrophoresis for rapid identification of monoclonal antibodies for routine application in hospital

mAbs are widely used in cancer therapy. Their compounding, performed just before their administration to patients, is executed in a production unit of the hospital. Identification of these drugs, individually prepared in bags for infusion before patient administration, is of paramount importance to detect potential mistakes during compounding stage. A fast and reliable analytical method based on CZE combined to a cationic capillary coating (hexadimethrine bromide) was developed for identification of the most widely used compounded therapeutic for cancer therapy (bevacizumab, cetuximab, rituximab, and trastuzumab). Considering the high structural and physico‐chemical similarities of these mAbs, an extensive optimization of the BGE composition has been performed. The addition of perchlorate ions and polysorbate in the BGE greatly increased the resolution. To validate the method, an internal standard was used and the relative migration times (RTm) were estimated. Very satisfactory RSDs of the RTm for rituximab (0.76%), cetuximab (0.46%), bevacizumab (0.31%), and trastuzumab (0.60%) were obtained. The intraday and interday RSD of the method were less than 0.32 and 1.3%, respectively for RTm. Significant differences between theses RTms have been demonstrated allowing mAbs identification. Finally, accurate mAbs identification has been demonstrated by a blind test.     

一种基于二喹啉甲酸- Cu+显色反应的毛细管电泳检测蛋白质的新方法

采用毛细管电泳法和蛋白质显色反应-二喹啉甲酸(BCA)法,结合微波辅助反应,在60 mmol/L硼酸盐缓冲液(pH 9.5)中,实现了对蛋白质的快速毛细管电泳分析检测。同时以β-环糊精为包合添加剂,实现了BCA-Cu+复合物和游离BCA的分离,从而在波长200 nm处以测定特征生成的BCA-Cu+复合物来间接检测蛋白质,其峰强度比直接检测蛋白质自身吸收的峰强度提高了2个数量级。对于转铁蛋白、蓖麻毒素,其线性范围分别为2～200 mg/L和2～100 mg/L,检出限分别为0.33和0.37 mg/L。将该方法成功地应用于第一届蓖麻毒素国际实验室间比对测试的部分样品,含量测定结果满意。     

Immunochemical method for sulfasalazine determination in human plasma

Sulfasalazine is an antibiotic used in the treatment of inflammatory bowel diseases. For the assessment of sulfasalazine in several biological matrices, an Enzyme-Linked Immunosorbent Assay (ELISA) method based on polyclonal antibodies was developed and characterized.The immunoassay showed a high sensitivity (IC₅₀ = 0.51 ng mL⁻¹) and specificity, a detection limit of 0.02 ng mL⁻¹ and a dynamic range of 0.06-3.75 ng mL⁻¹ (80-20% inhibition). The immunoassay performed well when it was applied to spiked plasma samples (from 0.5 to 2.0 ng mL⁻¹) previously cleaned up by protein precipitation with methanol. Recoveries ranged from 83 to 119%, with a mean value of 99% (CV = 13%).Since sulfasalazine remaining of a treatment reaches the systemic circulation in unchanged form, the immunoassay can be applied to the determination of this pharmaceutical in human plasma in order to facilitate the control of the patients through the application of personal doses.     

Use of MDLC-DIGE and LC-MS/MS to identify serum biomarkers for complete remission in patients with acute myeloid leukemia

The response criteria for complete remission (CR) in acute myeloid leukemia (AML) are currently based on morphology and blood cell counts. However, these criteria are insufficient to establish a diagnosis in cases with poor quality bone marrow (BM) samples demonstrating a loss of cellular morphology. We investigated whether the sera of patients contained biomarkers that indicate disease response status. First, we performed multidimensional liquid chromatography-differential gel electrophoresis (MDLC-DIGE) to generate protein profiles of two pooled, paired serum samples from patients who had achieved CR; one collected at diagnosis (PreCR) and the other collected after chemotherapy (CR). Then, with the biomarker candidates found, ELISA was carried out for individual PreCR and CR samples, and for other verification sets including nonremission (NR) patients and normal samples. We selected two proteins, complement factor H (CFH) and apolipoprotein H (ApoH), with dye (Cy) ratios showing greater than 2.0-fold differences between the pooled samples. ELISA showed that CFH and ApoH are useful for distinguishing between the recovered (CR and normal) and nonrecovered (PreCR, PreNR, and NR) states in AML (p <0.001). We successfully applied a protein profiling technology of MDLC-DIGE and LC-MS/MS to discover two biomarkers for CR which needs further validation for a clinical setting.     

Capillary zone electrophoresis method for the determination of famotidine and related impurities in pharmaceuticals

A simple and rapid capillary zone electrophoresis (CZE) method with UV detection has been developed for the determination of famotidine and its potential impurities in pharmaceutical formulations. The electrophoretic separation of these compounds was performed using a fused silica capillary and 37.5mmolL(-1) phosphate buffer pH 3.5 as the electrolyte. Under the optimised conditions, six impurities could be resolved from the famotidine peak in less than 7min. The calibration curves obtained for the seven compounds were linear over the concentration range investigated (from 1.5 to 78.5microg mL(-1)). The intra- and inter-day relative standard deviations for the migration times and corrected peak areas were less than 2% and 5%, respectively. The detection limits were found to be 0.09microg mL(-1) for famotidine, and from 0.1 to 0.62microg mL(-1) depending on the impurities. The method has been successfully applied to the determination of famotidine in commercial dosage forms.     

甲氧苄啶的毛细管电泳快速检测新方法

建立了毛细管电泳高频电导法测定药物和尿液中的甲氧苄啶。考察了各种条件对分离和检测的影响。以4.0 mmol/L HAc 体积分数10%甲醇(pH4.0)为电泳介质,分离电压20.0 kV,重力虹吸进样。在优化条件下,甲氧苄啶峰形良好,出峰时间小于6 min,线性范围为1.5~120.0μg/mL,检出限0.5μg/mL。该方法样品处理过程简单,可用于药物制剂的质量控制和临床检验。     

Capillary ion electrophoresis, an environmental method for the determination of anions in water

Capillary ion electrophoresis has recently been introduced as a new separations technique for the analysis of of inorganic anions. Among its many attributes are rapid, highly efficient separations with different selectives (compared to ion chromatography), simplicity, and economy.

This paper demonstrates the ability of capillary ion electrophoresis to analyze primary and secondary anionic contaminants as well as other ions of environmental concern in drinking water, groundwater, and wastewater. Analysis time is less than five minutes. A comparison of the data to ion chromatography shows excellent correlation.     

Validation of a capillary zone electrophoresis method for the comparative determination of etoricoxib in pharmaceutical formulations

A CZE method was developed and validated for the analysis of etoricoxib in pharmaceutical dosage forms, using prilocaine as an internal standard. The CZE method was carried out on a fused-silica capillary (50 microm id, effective length 40 cm). The BGE consisted of 25 mM tris-phosphate solution at pH 2.5. The capillary temperature was maintained at 35 degrees C, the applied voltage was 25 kV, the injection was performed using the pressure mode at 50 mbar for 5 s, with detection at 234 nm using a photodiode array detector. The method was linear in the range of 2-150 microg/mL (r(2) = 0.9999). The specificity and stability-indicating capability were proven through the degradation studies and showing also that there was no interference of the excipients of the formulation. The accuracy was 99.49% with RSD of 0.66%. The limits of quantitation and detection were 2 and 0.58 microg/mL, respectively. Moreover, method validation demonstrated acceptable results for the precision, sensitivity, and robustness. The proposed method was successfully applied for the quantitative analysis of etoricoxib pharmaceutical formulations, and the results compared to the HPLC and LC-MS/MS methods, showing nonsignificant difference (p >0.05).     

High performance capillary electrophoresis method for determination of ibuprofen enantiomers in human serum and urine

A direct and stereospecific capillary zone electrophoresis (CZE) method for quantification ibuprofen enantiomers in biological matrices: human serum and urine, has been developed. Chiral separation of the enantiomers of ibuprofen and (+)-S-indobufen [(+)-S-INDB, internal standard, IS] was obtained in an uncoated silica capillary filled with a background electrolyte (BGE), consisted of heptakis 2,3,6-tri-O-methyl-β-cyclodextrin (TM-β-CD) in buffer of pH 5.0. The complete enantioselective analysis of ibuprofen and its 1-hydroxy metabolite confirmed appropriate specificity of the method. The electrophoretic parameters: electroosmotic (μ_EOF) and electrophoretic (μ_ep) mobility and resolution factor (R_s) were determined. Extraction procedures with organic solvent and solid phase extraction (SPE) with C₁₈ stationary phase for isolation of enantiomers from biological fluids were compared. SPE method for further studies was chosen. Stereoselective extraction of IBP enantiomers from serum at basic pH has been discovered. Validation of the method was carried out. Calibration curves of ibuprofen enantiomers were linear in the range of 0.1-25.0 μg/ml in serum and of 0.5-250.0 μg/ml in urine. Recovery of both enantiomers from serum and urine amounted 74-86 and 90-98%, respectively. Intra- and inter-day measurement precision and accuracy were below 15%. Limits of detection for IBP enantiomers amounted 0.05 and 0.25 μg/ml in samples of serum and urine, respectively. Limit of quantitation was also estimated. IBP enantiomers proved to be stable following three freeze and thaw cycles and during storage in autosampler at ambient temperature. The validated methods enable pharmacokinetic studies of enantiomers in both media. The elaborated HPCE method can be alternative to HPLC.     

Influence of salt and acetonitrile on the capillary zone electrophoresis analysis of imatinib in plasma samples

A simple, sensitive, specific, and cost‐effective analytical methodology was developed for the analysis of human plasma samples spiked with imatinib by CZE with on‐line UV detection in the context of Therapeutic Drug Monitoring. Several analytical conditions such as the ionic strength (I) and the pH of the BGE composed of citric acid and ε‐amino caproic acid were studied in regards of the presence of sodium chloride (NaCl) in plasma samples (1% m/v). Computer simulations (Simul software) were used to confirm the experimental results and to understand imatinib electrophoretic behavior in the presence of NaCl. Furthermore, the advantages of adding ACN to the sample containing NaCl to combine efficient protein precipitation and on‐line CZE stacking of imatinib were demonstrated. LOD and LOQ values of 48 and 191 ng/mL were obtained from plasma sample supernatant after protein precipitation with ACN, which is much lower than mean imatinib plasma level observed for patients treated by imatinib mesylate (about 1000 ng/mL). Good linearity was obtained in the concentration range 191–5000 ng/mL (R² > 0.997). RSD of less than 1.68% and 2.60% (n = 6) for migration times and corrected peak areas, respectively, were observed at the LOQ.     

Optimization of a liquid-liquid extraction method for HPLC-DAD determination of penicillin-V in human plasma

Optimization of a liquid-liquid extraction procedure used for isolation of penicillin-V in human plasma samples and the subsequent HPLC-DAD analysis are reported. Some aspects related to the stability of penicillin-V in plasma and according to pH are also given. From seven tested solvents, the highest extraction yield was obtained with methyl-t-buthyl ether. Influence of the extraction parameters, such as pH of aqueous phase, sample/solvent volumetric ratio, and extraction duration, is discussed. HPLC separation was achieved on an Eclipse XDB-C8 column, using a mobile phase composed by aqueous phosphate buffer (pH=7.3), methanol and acetonitrile in the ratio 8:1:1. Quantitation limits of 50 ng/ml were obtained. The optimal sample preparation method and HPLC-DAD separations were used for a bioequivalence study, made on 36 volunteers.